Reactive oxygen species (ROS) play a significant role in the pathophysiology of cardiovascular disease. Though ROS have been conventionally regarded as harmful and toxic by-products of cellular aerobic metabolism, emerging evidence has suggested that ROS also function as crucial intracellular signaling molecules, capable of regulating cellular growth and hypertrophy. NADPH plays multiple roles in the regulation of ROS levels through its actions as a cofactor for antioxidant enzymes. NADPH also serves as a substrate for the generation of ROS by key enzymes that modulate cell growth/hypertrophy via ROS-dependent signaling pathways. In this context, the role of glucose-6-phosphate dehydrogenase (G6PD) and the pentose phosphate pathway (PPP) becomes extremely important. G6PD functions as the first and rate-limiting enzyme in the PPP, responsible for the generation of NADPH in a reaction coupled to the oxidation of glucose-6-phosphate. Although G6PD is ubiquitously expressed, the role of G6PD in regulating antioxidant defense and cell signaling pathways in the heart remains unknown. Our preliminary data suggest that inhibition of G6PD depletes antioxidant reserves and increases susceptibility to acute oxidative injury in cardiomyocytes, consistent with the role of G6PD as an antioxidant enzyme. Conversely, we have found that inhibition of G6PD also results in marked attenuation of mitogen-activated protein kinase ERK1/2 activation and cardiomyocyte hypertrophy following alpha1-adrenergic receptor stimulation, supporting the role of G6PD in modulating cell growth/hypertrophy. These observations led to our central hypothesis that G6PD, the rate-limiting enzyme in the pentose phosphate pathway, regulates cytosolic NADPH levels, and thereby (1) protects the heart from oxidative injury and (2) modulates myocardial growth/hypertrophy. We will utilize a multidisciplinary approach of in-vitro (isolated adult cardiomyocytes), ex-vivo (isolated Langendorff perfused hearts) and in-vivo (mouse models) methods to define the importance of the PPP and G6PD in the heart as well as explore its potential as a therapeutic target for the treatment of cardiovascular diseases associated with oxidative stress.